Methods and compositions for the treatment of wounds

ABSTRACT

Devices and methods for enhancing the healing of wounds, especially chronic wounds (e.g., diabetic wounds), involving the use of keratinocytes are described. Keratinocytes are grown on a transplantable solid support (e.g., collagen-coated beads), and the keratinocyte-coated solid support is placed in an enclosure. The enclosure, in turn, is placed in the wound for use as an interactive wound healing promoter.

The present Application is a Continuation of Ser. No. 09/338,413, filedJun. 22, 1999, which is a Continuation of Ser. No. 08/840,804, filedApr. 16, 1997, now U.S. Pat. No. 5,972,332.

FIELD OF THE INVENTION

The present invention relates generally to tissue healing andregeneration and, more particularly, to methods and systems for woundhealing.

BACKGROUND OF THE INVENTION

The primary goal in the treatment of wounds is to achieve wound closure.Open cutaneous wounds represent one major category of wounds and includeburn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers,and diabetic ulcers. Open cutaneous wounds routinely heal by a processwhich comprises six major components: i) inflammation, ii) fibroblastproliferation, iii) blood vessel proliferation, iv) connective tissuesynthesis v) epithelialization, and vi) wound contraction. Wound healingis impaired when these components, either individually or as a whole, donot function properly. Numerous factors can affect wound healing,including malnutrition, infection, pharmacological agents (e.g.,actinomycin and steroids), diabetes, and advanced age [see Hunt andGoodson in Current Surgical Diagnosis & Treatment (Way; Appleton &Lange), pp. 86-98 (1988)].

Wounds which do not readily heal can cause the subject considerablephysical, emotional, and social distress as well as great financialexpense [see, e.g., Richey et al., Annals of Plastic Surgery23(2):159-165 (1989)]. Indeed, wounds that fail to heal properly andbecome infected may require excision of the affected tissue. A number oftreatment modalities have been developed as scientists' basicunderstanding of wounds and wound healing mechanisms has progressed.

The most commonly used conventional modality to assist in wound healinginvolves the use of wound dressings. In the 1960s, a major breakthroughin wound care occurred when it was discovered that wound healing with amoist occlusive dressings was, generally speaking, more effective thanthe use of dry, non-occlusive dressings [Winter, Nature 193:293-94(1962)]. Today, numerous types of dressings are routinely used,including films (e.g., polyurethane films), hydrocolloids (hydrophiliccolloidal particles bound to polyurethane foam), hydrogels (cross-linkedpolymers containing about at least 60% water), foams (hydrophilic orhydrophobic), calcium alginates (nonwoven composites of fibers fromcalcium alginate), and cellophane (cellulose with a plasticizer) [Kannonand Garrett, Dermatol. Surg. 21:583-590 (1995); Davies, Burns 10:94(1983)]. Unfortunately, certain types of wounds (e.g., diabetic ulcers,pressure sores) and the wounds of certain subjects (e.g., recipients ofexogenous corticosteroids) do not heal in a timely manner (or at all)with the use of such dressings.

Several pharmaceutical modalities have also been utilized in an attemptto improve wound healing. For example, treatment regimens involving zincsulfate have been utilized by some practitioners. However, the efficacyof these regimens has been primarily attributed to their reversal of theeffects of sub-normal serum zinc levels (e.g., decreased host resistanceand altered intracellular bactericidal activity) [Riley, Am. Fam.Physician 24:107 (1981)]. While other vitamin and mineral deficiencieshave also been associated with decreased wound healing (e.g.,deficiencies of vitamins A, C and D; and calcium, magnesium, copper, andiron), there is no strong evidence that increasing the serum levels ofthese substances above their normal levels actually enhances woundhealing. Thus, except in very limited circumstances, the promotion ofwound healing with these agents has met with little success.

What is needed is a safe, effective, and interactive means for enhancingthe healing of chronic wounds. The means should be able to be usedwithout regard to the type of wound or the nature of the patientpopulation to which the subject belongs.

SUMMARY OF THE INVENTION

The present invention is directed at systems and methods for enhancingthe healing of wounds, especially chronic wounds (e.g., diabetic wounds,pressure sores), involving the use of cultured keratinocytes. In someembodiments, the invention contemplates the use of keratinocytes grownon a transplantable solid support. The present invention is not limitedby the nature of the solid support; indeed, the present inventioncontemplates the use of any three-dimensional support or matrix (e.g.,matrices comprised of glycosaminoglycans) to which keratinocytes willadhere, divide, and maintain their functional behaviors (e.g., healwounds).

In preferred embodiments, the solid support comprises collagen-coatedbeads. In particular embodiments, the collagen-coated beads are placedin an enclosure, compartment, bag, or similar barrier, said enclosurehaving pores, and the enclosure is then placed at the wound site for useas an interactive wound healing promoter. The present invention is notlimited by the nature of enclosure; however, in one embodiment, thepores are large enough to permit the cells from the beads to exit theenclosure into the wound, while in another embodiment, the pores are toosmall to permit cells from the beads to exit the enclosure, but largeenough to permit cellular factors to exit the enclosure or wound fluidcomponents to enter the enclosure. In certain embodiments, theenclosures are replaced every few days until the wound heals.

More particularly, the present invention contemplates a system for thetreatment of wounds, comprising a) keratinocytes on a solid support; andb) an enclosure, the enclosure housing the solid support. In someembodiments, the solid support comprises beads, and in furtherembodiments, the beads are macroporous. In still further embodiments,the beads are coated with an extracellular matrix (e.g., collagen).While the present invention is not limited to the nature of thekeratinocytes, in a preferred embodiment the keratinoctes are viable andgrowing.

In additional embodiments, the enclosure comprises a mesh material,having pores. In certain embodiments, the mesh material comprisespolyester. In one embodiment, the pores are large enough to permit thecells from the beads to exit the enclosure into the wound, while inanother embodiment, the pores are too small to permit cells from thebeads to exit the enclosure, but large enough to permit cellular factors(e.g., cytokines) to exit the enclosure or wound fluid components toenter the enclosure.

Moreover, in further embodiments, the enclosure comprises abiocompatible membrane. In additional embodiments, the enclosurecomprises means for removing the enclosure from a wound. In particularembodiments, the removal means comprises a handle or string attached tothe enclosure.

The present invention also contemplates a method for treating a wound,comprising a) providing: i) keratinocytes on a solid support, ii) anenclosure, and iii) a subject having a least one wound; b) placing thekeratinocyte-containing solid support into the enclosure so as toproduce a keratinocyte-containing enclosure; and c) positioning thekeratinocyte-containing enclosure in the wound of the subject underconditions such that the healing of the wound is promoted. Additionalembodiments further comprise, after step b) and prior to step c),sealing the enclosure to produce a sealed keratinocyte-containingenclosure. Finally, some embodiments further comprise step d), coveringthe wound containing the keratinocyte-containing enclosure with adressing.

DEFINITIONS

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

The term “wound” refers broadly to injuries to the skin and subcutaneoustissue initiated in different ways (e.g., pressure sores from extendedbed rest and wounds induced by trauma) and with varying characteristics.Wounds may be classified into one of four grades depending on the depthof the wound: i) Grade I: wounds limited to the epithelium; ii) GradeII: wounds extending into the dermis; iii) Grade III: wounds extendinginto the subcutaneous tissue; and iv) Grade IV (or full-thicknesswounds): wounds wherein bones are exposed (e.g., a bony pressure pointsuch as the greater trochanter or the sacrum). The term “partialthickness wound” refers to wounds that encompass Grades I-III; examplesof partial thickness wounds include burn wounds, pressure sores, venousstasis ulcers, and diabetic ulcers. The term “deep wound” is meant toinclude both Grade III and Grade IV wounds.

The term “chronic wound” refers to a wound that has not healed within 30days.

The phrase “positioning the enclosure in the wound” is intended to meancontacting some part of the wound with the enclosure. “Containing”includes, but is not limited to, bringing the enclosure proximate to thewound so as to bring the cells in fluidic communication with the wound.

The phrases “promote wound healing,” “enhance wound healing,” and thelike refer to either the induction of the formation of granulationtissue of wound contraction and/or the induction of epithelialization(i.e., the generation of new cells in the epithelium).

The phrase “wound fluid contents” refers to liquid associated with awound, as well as cells, cell factors, ions, macromolecules and proteinmaterial suspended Such liquid at the wound site.

The term “keratinocyte” refers to cells that produce keratin (ceratin),a scleroprotein or albuminoid. Generally speaking, keratinocytes arefound in the epidermis or from cell lines derived from keratinocytes(e.g., bacterial derived products).

The term “subject” refers to both humans and animals.

The terms “enclosure,” “compartment,” and the like refer broadly to anycontainer capable of confining a cell-coated solid support within adefined location while allowing cellular factors to exit the enclosureinto the wound and wound fluid contents to enter. In preferredembodiments, the enclosure is a sterile mesh pouch constructed of awoven, medical-grade polyester mesh. In one embodiment, the presentinvention contemplates a degradable enclosure (i.e., an enclosure thatbreaks down over time). In addition, the present invention contemplatesthe use of an enclosure constructed from membranes. Preferably, afterthe solid support containing cells (e.g., growing on the surface of thesurface of the solid support or within the solid support) is placedwithin the enclosure, the enclosure is sealed so as to prevent the solidsupport from exiting the enclosure. In one embodiment, the sealedenclosure further comprises a transport means for transporting cellularfactors (e.g., outside of the enclosure and into the wound). While thepresent invention is not limited to a particular transport means, thetransport means can include a means for applying pressure (e.g., apump).

The term “solid support” refers broadly to any support that allows forcell growth, including, but not limited to, microcarrier beads, gels,and culture plate inserts. Microcarrier beads suitable for use with thepresent invention are commercially-available from a number of sources,including Sigma, Pharmacia, and ICN. In preferred embodiments, thekeratinocytes are grown on collagen-coated beads (e.g., CYTOLINE 1™macroporous microcalTier beads (Pharmacia Biotech)). Culture plateinserts (i.e., cell support matrices that generally comprise a membranethat supports cell growth) are commercially available from, among othersources, Collaborative Biomedical Products, Costar, ICN, and Millipore.In preferred embodiments, the culture plate inserts comprise a permeablemicroporous membrane that allows free diffusion of ions andmacromolecules.

The term “transplantable solid support” refers to a solid supportcontaining cells (e.g., keratinocytes, referred to as a“keratinocyte-containing solid support”) that can be placed within anenclosure. The enclosure containing the cell-containing solid supportmay then be placed in a wound to promote wound healing.

The phrases “means for removing,” “removal means,” and the like referbroadly to any mechanism useful for assisting in the withdrawal of acell-containing enclosure from a wound (and/or the placement of thecell-containing enclosure within a wound). In some embodiments, theremoval means comprises a string, thread, cord, or the like that isattached to the enclosure; in preferred embodiments, the removal meansis attached to a grasp that can be used as a handle to assist in theplacement of the solid support-containing enclosure within the wound andits removal therefrom.

The term “dressing” refers broadly to any material applied to a woundfor protection, absorbance, drainage, etc. Numerous types of dressingsare commercially available, including films (e.g., polyurethane films),hydrocolloids (hydrophilic colloidal particles bound to polyurethanefoam), hydrogels (cross-linked polymers containing about at least 60%water), foams (hydrophilic or hydrophobic), calcium alginates (nonwovencomposites of fibers from calcium alginate), and cellophane (cellulosewith a plasticizer) [Kannon and Garrett, Dermatol. Surg. 21:583-590(1995); Davies, Burns 10:94 (1983)]. The present invention alsocontemplates the use of dressings impregnated with pharmacologicalcompounds (e.g., antibiotics).

The term “biocompatible” means that there is minimal (i.e., nosignificant difference is seen compared to a control), if any, effect onthe surroundings. For example, in some embodiments of the presentinvention, the enclosure comprises a biocompatible membrane; themembrane itself has a minimal effect on the cells of the solid support(i.e., it is non-toxic and compatible with keratinocyte growth) withinthe membrane and on the subject (i.e., it has no adverse impact on thesubject's health or the rate of wound healing) after the enclosure isplaced into a wound.

The term “extracellular matrix” refers broadly to material forsupporting cell growth. It is not intended that the present invention belimited by the particular material; the present invention contemplates awide variety of materials, including, but not limited to, material thatis distributed throughout the body of multicellular organisms such asglycoproteins, proteoglycans and complex carbohydrates. The presentinvention contemplates the use of a substratum of extracellular matrixwith the culture inserts on which the cells (e.g., keratinocytes) areplated. Although the present invention is not limited by the nature ofthe extracellular matrix, the preferred extracellular matrices includeMatrigel, Growth Factor Reduced Matrigel, fibrillar collagen, lamininn,fibronectin and collagen type IV. Collagen is the most preferredextracellular matrix for use with the present invention. However, thepresent invention is not limited to the use of collagen, nor to the useof solid supports that are commercially coated with collagen or otherextracellular matrices.

DESCRIPTION OF THE DRAWING

The FIGURE diagrammatically depicts one embodiment of a tea bagcontemplated for use with the cell-containing solid supports of thepresent invention. An enclosure, 1, is connects to a removal means, 2and 3, and contains solid support, 4. In this embodiment, the removalmeans comprises a string, 2, and a tab connected to the string, 3.

DESCRIPTION OF THE INVENTION

The present invention relates generally to tissue healing andregeneration and, more particularly, to methods and systems for woundhealing.

The invention involves the unique use of cultured cells to treat wounds,especially chronic wounds (e.g., diabetic wounds). In preferredembodiments, cultured keratinocytes grown on transplantable solidsupports are placed in a permeable enclosure; the enclosure is thenplaced in a wound. Though a precise understanding of how thecell-containing enclosure effects wound healing is not required in orderto practice the present invention, it is believed that the cells in theenclosure secrete certain factors that enhance wound healing. Theusefulness of the present invention has been demonstrated in athymicnude mice, an animal model routinely utilized in wound closure testing[see, e.g., Boyce et al., Surgery 110:866-76 (1991); Barbul et al.,Surgery 105:764-69 (1989); and Hansbrough et al., J. Burn Care Rehabil.14:485-94 (1993)].

The present invention is not limited by the nature of the cellsutilized. Examples of cells include, but are not limited to, the cellsset forth in Table I.

TABLE 1 CYTOCKINE, GROWTH WOUND FACTOR HEALING CELL TYPE TISSUEMADE/RESPONDS TO MATRIX INTERACTIONS POTENTIAL Fibroblast Dermis ViseralTGF-beta, PDGF, IGF, IL-1, Collagen type I, III, and IV Elastin,Fibroblast ⁺4 Organs FGF, CTGF Fibronectin, nidogen, SPARC, Osteonectin,Protenglycons, glucosamino-glycons, collagenases, gelatinase,stromelysin, TIMP, Thrombospondin Endothelial Blood Vessels FGF, VEGF,Endothelin, TIMP, GAG, Elastin, Laminin, Endothelial Cell ⁺4 Cell IGF,IL-1 Collagenase, Type IV Collagens Fibronectin Melanocyte Dermis IL-1,MSH No ECM Production Melanocyte ⁺1 Smooth Blood Vessels PGDG, IGF, EGF,FGF TIMP, GAG, Elastin, Laminin, Smooth Muscle Muscle Cell Collagenase,Collagens, Fibronectin Cell ⁺3 Fetal Fetal FGF, TGF-beta, PDGF, TIMP,GAG, Elastin, Laminin, Fetal Fibroblast ⁺3 Fibroblast Mesenchyma ILGF,IL-1, FGF Collagenase, Collagens, Fibronectin Epithelial Dermis FGF,TGF-alpha, TGF-beta TIMP, GAG, Elastin, Laminin, Epithelial Cell ⁺4 CellMucosa PDGF, IGF, IL-1 EGF, Collagenase, Collagen type IV, VI, VII, FGF,KGF IFN-gamma laminins, Fibronectin, epiligrin, nidogen, TNF-alpha, IL-1elastin, tenascin, thrombospondin, GAGs, alpha, activin proteoglycons,EMMPRIN, SPARC, uPA, PAI, collagenase, gelatinase, stromelysinABBREVIATION GLOSSARY Cytokine, Growth Factors Made/Responds To MatrixInteractions TGF Transforming Growth Factor TIMP Tissue Inhibitor ofMetalloproteinases PDGF Platelet Derived Growth Factor GAG GlucoseAminoglycons IGF Insulin-like Growth Factor SPARC Secreted ProteinAcidic and Rich in Cysteine IL Interleukin ECM Extracellular Matrix FGFFibroblast Growth Factor EMMPRIN Extracellular Matrix MetalloproteinaseInducer CTGF Connective Tissue Growth Factor uPA Urokinase TypePlasminogen Activator VEGF Vascular Endothelial Growth Factor PAIPlasminogen Activator Inhibitor MSH Melanocyte Stimulating Hormone EGFEpidermal Growth Factor KGF Kerotinocyte Growth Factor IFN Interferon

I. SOURCES OF KERATINOCYTES

The present invention is not limited by the source of the keratinocytes.In some preferred embodiments, the cells are obtained from living donorsundergoing breast operations; prior to their use, the cells obtainedfrom the donors are archived for at least six months, after which theyare tested for the presence of viruses (e.g., hepatitis virus). In otherpreferred embodiments, the cells are cadaveric in origin. After thecells have been harvested from the cadaver, they are screened forviruses and other microbes prior to use.

Generally speaking, the keratinocytes contemplated for use with thepresent invention are primary cultured cells (i.e., the cells are notderived from cell lines) or are cells that have been transfected anddeveloped into a keratinocyte derived cell line.

Example 1 in the Experimental section illustrates one embodiment of howkeratinocytes may be isolated and processed for use with the presentinvention. However, it should be noted that the present invention is notlimited to primary cultured cells.

Moreover, the present invention contemplates the use of cells that havesimilar characteristics to keratinocytes (e.g., cells that secretegrowth factors, cytokines or keratin, whose behavior the cells utilizeto promote wound healing). These cells may be derived, for example, fromcells that are not keratinocytic in origin but have been modified byrecombinant techniques.

II. GROWTH OF CELLS ON SOLID SUPPORTS

The cells contemplated for use with the present invention (e.g.,keratinocytes) are grown on transplantable solid supports. The presentinvention contemplates the growth of keratinocytes on solid supports,including protein-coated solid surfaces, as has been described in theart. For example, Gilchrest et al. [Cell Bio Int. Rep. 4:1009 (1980)]describe the growth of keratinocytes on fibronectin-coated plates in theabsence of a 3T3 monolayer, while Schafer et al. [Exp. Cell. Res.183:112 (1989)] describe a study of keratinocytes on floating collagengels. Furthermore, Cook and Buskirk [In Vitro Cell Dev. Biol. 31:132(1995)] describe the growth of keratinocytes on a variety of matrices,including microporous membranes coated with collagen.

The present invention is not limited by the nature of the solid support.Indeed, the methods of the present invention may be practiced inconjunction with any support that allows for cell growth, including, butnot limited to, microcarrier beads, gels, and culture plate inserts.When microcarrier beads are desired, suitable beads arecommercially-available from a number of sources; for example, Sigmasells both collagen- and gelatin-coated beads, Pharmacia sellsdextran-based beads, and ICN advertises collagen beads. In preferredembodiments, the keratinocytes are grown on collagen-coated beads (e.g.,CYTOLINE 1™ macroporous microcarrier beads (Pharmacia Biotech)).

Furthermore, culture plate inserts (i.e., cell support matrices thatgenerally comprise a membrane that supports cell growth) arecommercially available from, among other sources, CollaborativeBiomedical Products, Costar, ICN, and Millipore. Such inserts frequentlycomprise polyethylene terephthalate, polycarbonate, TEFLON® (Gore), andmixed cellulose esters. In particular embodiments, the culture plateinserts comprise a permeable microporous membrane that allows freediffusion of ions and macromolecules.

As indicated above, the present invention contemplates the use oftransplantable solid supports. More specifically, the present inventioncontemplates the application of keratinocyte-coated solid supports,housed in an enclosure, to wounds. The use of cell-coated transplantablesolid supports for application to wounds has been described in the art.For example, Hansbrough et al. [J. Am. Med. Assoc. 262:2125 (1989)]describe collagen-glycosaminoglycan membranes covered with keratinocytesfor wound application. [See also, Cooper et al., J. Surg. Res. 48:528(1990); Ronfard et al. Burns 17:181 (1991); Tinois et al., Exp. CellRes. 193:310 (1991); and Nanchahal and Ward, Brit. J. Plas. Surg. 45:354(1992)]. However, the enclosure of keratinocyte-coated solid supportshas not been reported.

Generally speaking, growth of keratinocytes and other“anchorage-dependent” cells requires attachment to a surface andspreading out in order to grow. Conventionally, such cells have beencultured on the walls of non-agitated vessels (e.g., tissue cultureflasks) and roller bottles [U.S. Pat. No. 5,512,474 to Clapper et al.,hereby incorporated by reference]. Though not limited by the manner inwhich the keratinocytes are grown on the solid supports, the presentinvention contemplates the use of these conventional techniques forgrowing keratinocytes on solid supports (see Example 1).

Other techniques for culturing solid support-bound keratinocytes arecontemplated for use with the present invention. In some embodiments,the present invention contemplates the use of bioreactors for cellgrowth [see U.S. Pat. No. 5,459,069 to Palsson et al. and U.S. Pat. No.5,563,068 to Zhang et al., both hereby incorporated by reference]. Somebioreactors utilize hollow fiber systems. Frequently, bundles ofparallel fibers are enclosed in an outer compartment; cells are grown onthe outside surface of the fibers, while nutrient- and gas-enrichedmedium flows through the center of the hollow fibers, nourishing thecells [see, e.g., U.S. Pat. No. 5,512,474 to Clapper et al.].

In addition, bioreactors utilizing microcarriers (e.g., DEAE-derivativeddextran beads) can be used in conjunction with the present invention. Inpreferred embodiments, cell adhesion proteins like collagen,fibronectin, and laminin are used to anchor the cells to the solidsupport; collagen is the most preferred cell adhesion protein.Microcarriers may also incorporate an ionic charge to assist in cellattachment to the microcarrier. Frequently, the microcarriers are porousbeads that are sufficiently large to allow cells to migrate and grow inthe interior of the bead [see U.S. Pat. No. 5,512,474 to Clapper etal.].

In a particularly preferred embodiment, keratinocytes are supported on arigid support matrix (a semipermeable membrane) which allows for celladherence and growth. The cells form a dense, three-dimensional arraywith large surface area which enhances modification of the fluid phasebathing the cells; the cell-populated matrix is constantly exposed towound fluid components which diffuse into the reactor. The fluid can bemodified and/or the cells can secrete mediators into the fluid tooptimize the wound environment.

III. ENCLOSURES

The present invention contemplates the placement of keratinocyte-coatedcollagen beads in an enclosure, which, in turn, is placed in a wound. Inpreferred embodiments, the enclosure is a sterile mesh pouch constructedof a woven, medical-grade polyester mesh. Though not limited to meshmaterials manufactured by any particular company, Tetko, Inc. and Saaticurrently manufacture mesh materials suitable for use with the presentinvention.

Of course, other suitable materials (e.g., nylon) may also be used andare within the scope of the present invention. Indeed, any material thatexhibits biocompatibility when placed within a wound may be used withpresent invention. In addition, the present invention contemplates theuse of an enclosure constructed from membranes, including the membranessold commercially by Gelman Sciences and Millipore.

In a preferred embodiment, the enclosures are assembled as pocket-likecontainers with four edges and two surfaces. These containers may bemanufactured in one of several ways. For example, the enclosure may becreated by welding (i.e., uniting to create a seal) two pieces ofmaterial (of approximately equal dimensions) together on three edges.The fourth edge is left open to allow filling of the enclosure with thekeratinocyte-coated collagen beads.

In an alternative embodiment, the enclosure may be manufactured from onepiece of material by first folding that piece of material back ontoitself. The region where the material overlaps itself may then bewelded, resulting in the formation of a cylindrical tube. Thereafter, apocket can be fonned by welding closed one of the open ends of thecylinder, leaving the other end open for filling with thekeratinocyte-coated collagen beads; this enclosure design has theadvantage of requiring one less weld.

The present invention is not limited to enclosures assembled asfour-edged pockets nor is the invention limited to the techniques ofconstructing the enclosures disclosed above. For example, trapezoidal orcircular enclosures may also be used in conjunction with the presentinvention.

For the assembly of the enclosures, the present invention contemplatesthe use of a variety of sealing techniques, including ultrasonic weldingor heat welding. The technique of ultrasonic welding is well-known inthe medical device-manufacturing art [see, e.g., U.S. Pat. Nos.4,576,715 and 5,269,917, hereby incorporated by reference]. The presentinvention is not limited to a particular welding/sealing technique;indeed, any suitable sealing technique may be used with the presentinvention, including but not limited to ultrasonic, radiofrequency,heat, and impulse sealing.

In those embodiments comprising a mesh enclosure, the present inventionis not limited by the pore size of the mesh. However, it should be notedthat extremely small pores may retard or preclude the movement ofmaterials out of the enclosure. The preferred range of pore sizes isfrom about 10 microns to about 300 microns. Likewise, if a membrane isused, the membrane must be permeable to the extent that it allow thecell factors to cross the membrane into the wound.

In preferred embodiments, the solid support-containing enclosures of thepresent invention are configured like tea-bags (see FIGURE). That is,one end of a handle (3) (e.g., a biocompatible nylon material or excessfrom a heat seal) is attached to the enclosure (1) housing the solidsupport (4), while the other end of the string is attached to a grasp(2). The grasp (2) is used as a “handle” to assist in the placement ofthe solid support-containing enclosure within the wound and its removaltherefrom. The present invention is not limited by the material used toconstruct the grasp; in preferred embodiments, the grasp (2) comprises amedical grade polyester material. Generally speaking, the grasp (2) istaped to the subject's skin at a site external to the wound. The solidsupport (4) has cells (e.g., keratinocytes) attached; it is preferredthat such cells are viable.

IV. TRANSFER OF CELL FACTORS

Following placement of the enclosure within the wound, the cell factors(e.g., growth factors like epidermal growth factor, cytokines, PGDF,insulin like growth factor, TGF-beta, keratinocyte growth factorcytokine, TNF, chemokines, chemotactic peptides, tissue inhibitors ofmetalloprotcinases, etc.) excreted from the keratinocytes pass throughthe enclosure and into the wound. The inventors of the present inventionhave found that it is not necessary for the keratinocytes to be indirect contact with the wound. Though an understanding of why suchindirect contact is sufficient for wound healing is not required inorder to practice the present invention, it is believed that the donorkeratinocytes (i.e., those contained within the enclosure) create afavorable environment for growth of the keratinocytes present in thewound of the subject. Thus, the keratinocytes from the healed wound siteare thought to be of recipient, rather than donor, origin [see Van derMerve et al., Burns 16:193 (1990)]. In addition, the keratinocytes mayactively modify wound fluid characteristics or components (e.g.,modulating protolytic activity to optimize the wound environment.

The inventors of the present invention discovered empirically thatplacement of keratinocyte-coated solid supports within the enclosures(described above) resulted in good “take” of keratinocytes in deepwounds; in comparison, researchers previously reported less than idealtake in deeper wounds [Van der Merve et al., Burns 16:193 (1990)] whenother techniques were used.

The inventors have found that the use of the present invention inconjunction with standard wound dressing materials does not adverselyaffect the ability to modify the wound environment. For example, afterplacing the keratinocyte-containing enclosures within a wound, theenclosures can itself be covered with occlusive dressings such ashydrogels, foams, calcium alginates, hydrocolloids, and films. Example 2of the Experimental section addresses an embodiment wherein akeratinocyte-containing enclosure is covered by a wound dressing.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (Molar); mM (millimolar); μM (micromolar); g(grams); mg (milligrams); μg (micrograms); kg (kilograms); L (liters);mL (milliliters); dL (deciliters); μL (microliters); cm (centimeters);mm (millimeters); μm (micrometers); nm (nanometers); h and hr (hours);min. (minutes); s and sec. (seconds); FDA (United States Food and DrugAdministration); AET, Inc. (Middletown, Del.); Abbott (AbbottLaboratories, Chicago, Ill.); American International Electric Co. (SantaFe Springs, Calif.); Collaborative Biomedical Products (Bedford, Mass.);Gelman Sciences (Ann Arbor, Mich.); ICN (ICN Biomedicals, Inc., CostaMesa, Calif.); Jackson Labs (Bar harbor, Me.); Labelon Corp.(Canadaigua, N.Y.); Labline (Melrose Park, Ill.); MallinckrodtVeterinary (St Louis, Mo.); Millipore (Millford, Mass.); PharmaciaBiotech (Uppsala, Sweden); Richard-Allen, Inc. (Richland, Mich.); Saati(Stamford, Conn.); Sigma (St. Louis, Mo.); Tetko, Inc. (Depew, N.Y.); 3MHealthcare (St. Paul, Minn.); and Thunderware (Orinda, Calif.).

EXAMPLE 1

The experiments of this example demonstrate that human culturekeratinocytes grown on macroporous microcarriers and contained in aporous enclosure improve healing in surgically created wounds in mice.

A. Experimental Methodology

Preparation Of Human Keratinocytes

Isolation and Growing of Human keratinocytes

Human keratinocytes (AATB certified; University of Michigan culturedkeratinocyte program) were isolated at The University of MichiganBurn/Trauma Unit from split thickness skin.

Trypsinization of the split thickness skin was effected as follows. Theskin was placed dermis-side down in 150 mm Petri dishes. The pieces werecut into smaller pieces (about 2 cm×about 0.3 cm) and were soaked in asterile solution of 30 mM HEPES, 10 mM glucose, 3 mM KCl, 130 mM NaCl, 1mM Na₂HPO₄ buffer, pH 7.4 containing 50 units of Penicillin and 50 μgStreptomycin (Sigma, P-0906). After soaking for 1-2 hr at 4° C. thebuffer was aspirated off, and 0.09% trypsin (Sigma, Type IX) in aPenicillin and Streptomycin buffer was added to the dishes containingthe skin tissue.

After trypsinizing overnight at room temperature, the enzyme solutionwas aspirated off, and complete MCDB 153 (Gibco, Grand Island, N.Y.)medium containing trypsin inhibitor was added to the skin pieces.Complete MCDB 153 medium was made by supplementing basic MCDB 153(Gibco, Grand Island, N.Y.) medium, prepared as described by Boyce andHam [“Normal human epidermal keratinocytes,” In In Vitro Models forCancer Research (Weber and Sekely, eds.) CRC Press, Boca Raton, Fla.,pp. 245-274 (1985)], with 0.6 μM (0.218 μ/mL) hydrocortisone, 5 ng/mL,epideimal growth factor, 5 μg/mL insulin, 6 % bovine pituitary extract,and 0.15 mM CaCl₂.

The dermis was separated from the epidermis, and the epidermal basalcells were gently scraped off both segments of the skin. The cellsuspension was pooled into 50 mL conical centrifugation tubes, gentlycentrifuged at room temperature, and resuspended in 50 mL of completemedium plus 2% chelated serum.

The cells were counted using a hemacytometer, and 20×10⁶ cells wereplated into a T-75 Corning Plastic flasks and grown at 37° C. with 5%CO₂ gassing, using a humidified incubator. After 3 days, the used growthmedium was removed and complete MCDB 153 without serum was added. Thecells were fed every other day.

The cells were passaged during log phase of growth. Thereafter, thecells were trypsinized using 0.025% trypsin (type IX) plus 0.01% EDTA inthe HEPES buffer. The monolayers were washed with the buffer twice, then2-3 mL of freshly-made enzyme solution (or frozen aliquot) were added.After 1 mm. at 37° C., the enzyme solution was gently aspirated off, andthe cells were placed in flasks at 37° C. for 2-3 min. until the cellsheets came off the bottom with gentle tapping of the flask. The mediawas neutralized with 1-3 mL of MCDB 153 medium plus 0.03% trypsininhibitor (Sigma). The cells were counted, centrifuged, and plated 0.5to 1.0×10⁶ cells per T-75 flask. Cells were passaged 3 to 4 times.

CYTOLINE 1™ Bead Wash:

Five grams of CYTOLINE 1™ macroporous microcarrier beads (PharmaciaBiotech) were autoclaved for 10 min. in 40 mL Milli Q water (Millipore,Bedford, Mass.) in a 125 ml Erlenmeyer flask. Following the autoclavingprocedures, the beads were cooled and the water was aspirated. The beadswere re-suspended in 40 mL Milli Q water, and were then agitated atmoderate speed on a Labline orbital shaker for 10 min. The water wasagain aspirated, and a final washing with 40 mL Milli Q water wasperformed.

The beads were transferred into a 50 mL conical culture tube, the waterwas aspirated, and 30 ml 0.1 N NaOH were added. The beads were incubatedat room temperature overnight. The NaOH solution was aspirated off thebeads, and the beads were resuspended in 50 mL Milli Q water. Thealiquot was transferred to a 125 Erlenmeyer flask and shaken at moderatespeed for ten minutes. The Milli Q water was aspirated off the beads,and the beads were resuspended in Milli Q water; thisaspiration/resuspension procedure was repeated a total of five times.The pH was neutral (i.e., less than 8), as measured with pH paper.

The beads were aspirated and resuspended in 40 mL PBS without Mg²⁺ andCa²⁺, and autoclaved 30 min. at 121° C.

Growth of Keratinocytes on CYTOLINE 1™ Beads:

A slurry containing 10 mL of PBS solution and 5 g of beads (contained ina 50 mL sterile conical centrifuge tube) was autoclaved as describedabove. The PBS was decanted, and 50 mL of MCDB 153 complete medium wasadded to the beads. The cells were conditioned in the medium at 37° C.with 5% CO₂ gas for 48 hours.

The medium was decanted, and the beads were transferred into a separate50 mL sterile centrifuge tube. Ten-to-15 mL of medium were added, andthe suspension was centrifuged at 1000 rpm for 3 min. The medium wasagain decanted, and 30×10⁶ breast cells (from a living donor passage 1,never frozen) were added. After gently agitating the cells with thebeads for 5 minutes, the cells and beads were poured into a 250 mL glassroller bottle and 50 mL of medium was added; this was performed using afermentor-agitated growth system.

As a toxicity assay, 5 mL of cells and beads were removed from the glassroller bottle and grown in a T-25 flask to determine the growth of thecells on the plastic bottom of the flask in the presence of the beads.The roller bottle was incubated overnight at 37° C., after which 100 mLadditional medium was added to the roller bottle and the rotation of theroller bottle was initiated (rotation rate=one turn/15 sec.).

To feed the cells, an aliquot of medium was removed and replaced byfresh medium, adjusted to the correct ph with CO₂ gassing. The cellswere fed every 48 hours.

Experimental Design

An eight-day animal trial was conducted with two groups of ten animalseach. The wound dressings (see below) were changed every other daystarting on day 0. Wound area measurements and photographs were obtainedat days 0, 2, 4, 6, and 8.

All surgical procedures were performed under sterile conditions inside alaminar flow hood. Five-week old, female Nu/J mice (Jackson labs) wereused. Nu/J mice contain a recessive mutation found on chromosome 11 andare athymic (T-cell deficient). The mice have a reduced lymphocyte countcomprised almost entirely of B-cells, a normal IgM response tothymus-independent antigens, a poor response to thymus antigens,increased macrophage and NK cell activity, and increased susceptibilityto infection. Nu/J mice do not reject allogeneic and xenogeneic skin andtumor grafts.

The mice were anesthetized with metofane (Mallinckrodt Veterinary) andprepped with ethanol. Using fine surgical scissors, a full thicknesssurgical wound approximately 80 mm² in area was created on the backs ofthe mice (the depth of the wound could be measure through the panniculuscarnosis, but mouse skin is so thin so it was not used as an indicatorhere). The wound dressings (see below) were secured to the cephalad endof the wound with a surgical staple. Thereafter, each mouse was returnedto its biohazard containment cage.

On days 2, 4, 6 and 8, the animals were returned to the laminar flowhood for removal of the staple and replacement of the bag. The animalswere lightly restrained while area and photographic measurements wereobtained (described below). The dressing was replaced and secured; alldressing changes were performed using sterile technique without generalanesthesia.

Wound Dressing

The wounds were dressed either with human cultured keratinocytes grownon beads (keratinocytes/beads) in a DELNET™ bag (P530 Natural; AET,Inc.) or a DELNET™ bag alone (P530 Natural; AET, Inc.); the DELNET™ bagswere approximately square (about 23 mm×25 mm). The seams of the bagswere prepared with an Impulse heat sealing unit (American InternationalElectric Co.). Prior to application on the mice, the DELNET™ bags weregas sterilized with ethylene oxide and placed in a sterile package. ABANDAID™ (3M Healthcare) covered the DELNET™ bags and was secured withsurgical staples (Richard-Allen, Inc). The bag was stapled to thebandaid, and the bandaid was stapled to the mouse.

The bag and bead assembly was performed in a tissue culture hood. Insidea laminar flow hood, the keratinocytes/bead suspension was transferredto the DELNET™ bag with a glass pipet. Approximately 250 μL of thekeratinocyte/bead suspension was placed in the bag. After the beads wereloaded into the bag, the final seam was made with a surgical needleholder heated in a glass bead sterilizer. The DELNET™ bag containing thekeratinocytes/bead suspension is referred to as “beads/bag,” while theDELNET™ bag without the beads is referred to as “bag.” The bags andbeads/bags were placed in the complete MCDB 153 medium described aboveafter they were loaded and heat scaled.

Measurement of Wound Area

Total area of mouse wounds was performed as previously described[Schwarz et al., Wound Repair and Regeneration 3:204-212 (1995)].Briefly, the area of the wound was traced on transparency film (Apollo,Ronkonkoma, N.Y.) with a fine marker. The transparency film wasphotocopied onto plain paper and subsequently scanned into a PIC filewith a Lightning Scan Pro 256 hand scanner (Thunderware). Tissue areawas calculated with non-rectangular area analysis used by NIH image1.58, and the data was expressed as millimeters squared. Mean andstandard deviation were calculated using Statworks software (astatistically significant difference was p<0.05).

B. Experimental Results

Table 2 presents wound tissue area (mm²) at baseline (day 0) and at days2, 4, 6, and 8 for each mouse which received bags containingkeratinocyte-coated beads (beads/bags); the reduction in size of thewound as a percentage of the original wound size for each mouse is alsoset forth. Analogous data for the mice that received bags alone ispresented in Table 3.

Table 4 presents the cumulative data for i) the beads/bags mice and ii)the bags only mice.

TABLE 2 Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 mm² % Smaller mm² %Smaller mm² % Smaller mm² % Smaller mm² % Smaller Day 0 117.0 66.2989.89 69.9 103.8 2 2 Day 2 92.44 21 90.87 0 63.13 30 51.34 27 180.5 0 6Day 4 70.31 40 68.81 0 31.6 65 40.65 42 85.68 17 Day 6 80.87 31 54.21 1832.73 64 39.24 44 89.76 14 Day 8 50.05 57 37.18 44 26.73 70 31.95 5458.58 44 Mouse 6 Mouse 7 Mouse 8 Mouse 9 Mouse 10 mm² % Smaller mm² %Smaller mm² % Smaller mm² % Smaller mm² % Smaller Day 0 61.61 58.8180.08 78.02 65.5 Day 2 40.97 34 49.84 15 111.3 0 103.4 0 79.92 0 1 8 Day4 28.5 54 21.11 64 77.31 3 48.44 38 55.03 16 Day 6 23.53 62 15.89 7361.27 23 41.87 46 60.73 7 Day 8 10.31 83 12.48 79 29.55 63 33.69 5757.29 13

TABLE 3 Mouse 11 Mouse 12 Mouse 13 Mouse 14 Mouse 15 mm² % Smaller mm² %Smaller mm² % Smaller mm² % Smaller mm² % Smaller Day 0 49.58 87.1640.29 90.81 55.23 Day 2 71.23 0 160.2 0 45.5 0 201.1 0 61.55 0 3 5 Day 451.6 0 78.68 10 33.21 18 156.4 0 52.19 6 2 Day 6 26.48 47 52.24 40 27.9531 92.19 0 36.61 34 Day 8 33.84 32 52.4 40 15.19 62 59.15 35 30.61 45Mouse 16 Mouse 17 Mouse 18 Mouse 19 Mouse 20 mm² % Smaller mm² % Smallermm² % Smaller mm² % Smaller mm² % Smaller Day 0 120.4 59.11 120.1 78.2955.44 2 5 Day 2 90.26 25 151.4 0 151.4 0 114.1 0 89.89 0 8 Day 4 59.8450 124.8 0 124.8 0 138.7 0 66.58 0 1 1 3 Day 6 47.11 61 92.94 23 92.9423 92.39 0 59.6 0 Day 8 26.95 78 90.71 35 78.32 35 65.52 16 38.79 30

TABLE 4 Day 0 Size (mm²) Day 2 Day 4 Day 6 Day 8 Beads/ Bags Beads/ BagsBeads/ Bags Beads/ Bags Beads/ Bags Bags Only Bags Only Bags Only BagsOnly Bags Only Mean 79.09 75.64 12.7 2.5 33.9 8.4 38.2 26.6 56.4 37.3Std Deviation 19.2 28.64 14.29 7.9 23.82 15.83 23.06 22.62 20.12 21.78Significance (<0.05) p < 0.027 p < 0.008 p < 0.19 p < 0.05

As indicated by the data in Tables 2-4, the beads/bags showed astatistically significant difference in wound healing (i.e., a reductionin wound area) at day 2 compared to the bags alone (see Table 4,p<0.027). At day 4, the beads/bag (Table 2 1) treated mouse wounds had asignificant reduction in wound area compared to the mouse wounds in thebags alone (Table 3), as indicated by the significance level (p<0.008)in Table 4. At day 6, there was no significant difference in woundhealing between the two groups (see Table 3, p<0.16). However, at day 8,there was again a statistically significant reduction in the wound areain the beads/bag group (Table 2) compared to the bags alone group (Table3) (see Table 4, p<0.05).

The experiments of this example show that cultured human keratinocytesgrown on a macroporous microcarriers (beads/bag) promote wound healing.The mouse model used is predicative that human keratinocytes grown on amacroporous microcarriers contained in bags will enhance wound healingin humans.

EXAMPLE 2

The experiments of this example demonstrate that human culturekeratinocytes grown on macroporous microcarriers and contained in aporous enclosure that is then covered with a wound dressing materialimprove healing in surgically created wounds in mice.

A. Experimental Methodology

The experiments of this example were performed as described in Example1, with the following exceptions. The group of mice that received thekeratinocyte-coatcd CYTOLINE 1™ macroporous microcarrier beads(Pharmacia Biotech) (i.e., the beads/bags group) comprised five animals,while the group that received only the bags (i.e., the bags only group)comprised four animals. (They are labelled 2 to 5 because Mouse 1expired during anesthesia.) In this example the bags from both thebeads/bags group and the bags only group were covered with apolyurethane film dressing (TEGADERM™, 3M Healthcare, St. Paul, Minn.)with a cellophane product.

More specifically, the wounds were dressed either with human culturedkeratinocytes grown on beads (keratinocytes/beads) in a DELNET™ bag(P530 Natural; AET, Inc.) or a DELNET™ bag alone (P530 Natural; AET,Inc.). Thereafter, the bags were covered with a TEGADERM™ dressingwhich, in turn, was covered with a BANDAID™ (3M Healthcare). The bagswere stapled to the mouse.

B. Experimental Results

Table 5 presents wound tissue area (mm²) at baseline (day 0) and at days2, 4, 6, and 8 for each mouse which received bags containingkeratinocyte-coated beads (beads/bags); the reduction in size of thewound as a percentage of the original wound size for each mouse is alsoset forth. Analogous data for the mice that received bags alone ispresented in Table 6.

Table 7 presents the cumulative data for i) the beads/bags mice and ii)the bags only mice.

TABLE 5 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6 mm² % Smaller mm² %Smaller mm² % Smaller mm² % Smaller mm² % Smaller Day 0 182.4 132.8155.5 173.7 164.2 1 8 3 6 4 Day 2 162.4 11 168.5 0 157.0 0 190.5 0 216.60 3 7 2 1 6 Day 4 136.7 25 122.3 8 96.69 38 164.7 6 136.7 17 3 7 2 Day 674.71 59 89.91 33 49.66 68 25 86 25 85 Day 8 13.19 93 10.78 92 4.59 9718.24 90 27.87 83

TABLE 6 Mouse 7 Mouse 8 Mouse 9 Mouse 10 mm² % Smaller mm² % Smaller mm²% Smaller mm² % Smaller Day 0 134.14 198.31 124.1 229.96 Day 2 151.4 0170.21 0 166.42 0 203.36 0 Day 4 206.34 0 204.39 0 160.69 0 193.03 16Day 6 112.29 22 121.92 39 117.33 5 64.92 22 Day 8 16.86 88 53.45 7361.36 51 59.3 74

TABLE 7 Day 0 Size (mm²) Day 2 Day 4 Day 6 Day 8 Cell/ Bags Beads/ BagsBeads/ Bags Beads/ Bags Beads/ Bags Bags Only Bags Only Bags Only BagsOnly Bags Only Mean 155.76 171.16 2.2 0 18.8 0 66.2 22 91 71.5 StdDeviation 22.47 50.9 4.9 0 13.1 0 21.8 13.8 5.1 15.2 Significance(<0.05) p < 0.407 p < 0.026 p < 0.010 p < 0.030

As indicated by the data in Tables 5-7, the beads/bags demonstrated astatistically significant difference in wound healing (i.e., a reductionin wound area) at day 4 compared to the bags alone (see Table 7,p<0.026). The statistically significant difference in wound healingbetween the two groups was maintained on days 6 and 8 (p<0.010 andp<0.030, respectively).

Comparison of the data in Table 7 to that in Table 4 (Example 1)indicates that the wound dressings without TIGADERM™ begin to contractearlier than those with TEGADERM™. More specifically, the wounds of thebeads/bags animals treated without TEGADERM™ were 12.7% smaller by day 2and 33.9% smaller by day 4, while the wounds of the beads/bags animalstreated with TEGADERM™ were 2.2% and 18.8% smaller on the same days.However, the size of the wounds of the beads/bags animals treated withTEGADERM™ became smaller than those treated without TEGADERM™ on days 6and 8. While an understanding of the mechanism for this effect is notrequired in order to practice the present invention, it is believed tobe due, in part, to the ability of the TEGADERM™ to keep the woundsmoist.

The experiments of this example indicate that the systems and methods ofthe present invention can be practiced in combination with conventionalwound healing means and procedures.

Based upon the preceding discussion and experimental materials, itshould be clear that the present invention provides effective andefficient systems and methods for wound healing, especially healing ofchronic wounds. The devices and methods may be used alone or incombination with other means traditionally employed in wound healing.

We claim:
 1. An enclosure configured for housing keratinocytes on asolid support, wherein said enclosure comprises mesh material havingpores, said pores being too small to permit said keratinocytes on saidsolid support to cross said mesh material.
 2. The enclosure of claim 1,wherein said enclosure is sealable.
 3. The enclosure of claim 1, whereinsaid solid support comprises beads.
 4. The enclosure of claim 3, whereinsaid beads comprise macroporous beads.
 5. The enclosure of claim 4,wherein said macroporous beads comprise a collagen coating.
 6. A systemfor the treatment of wounds, comprising: a) keratinocytes on a solidsupport, b) an enclosure configured for housing said solid support,wherein said enclosure comprises mesh material having pores, said poresbeing too small to permit said keratinocytes on said solid support tocross said mesh material.
 7. The system of claim 6, wherein saidenclosure is sealable.
 8. The system of claim 6, wherein said solidsupport comprises beads.
 9. The system of claim 8, wherein said beadscomprise macroporous beads.
 10. The system of claim 9, wherein saidmacroporous beads comprise a collagen coating.
 11. A method forproducing a scaled keratinocyte-containing enclosure for promoting woundhealing, comprising; a) providing; i) keratinocytes on a solid support,ii) an enclosure configured for housing said solid support, wherein saidenclosure comprises mesh material having pores, said pores being toosmall to permit said keratinocytes on said solid support to cross saidmesh material; and b) sealing said enclosure to produce a sealedkeratinocyte-containing enclosure, wherein said sealedkeratinocyte-containing enclosure is configured for promoting woundhealing in a wound.
 12. The method of claim 11, wherein said solidsupport comprises beads.
 13. The method of claim 12, wherein said beadscomprise macroporous beads.
 14. The method of claim 13, wherein saidmacroporous beads comprise a collagen coating.
 15. The method of claim11, wherein said sealing is accomplished by ultrasonic welding.
 16. Themethod of claim 11, wherein said sealing is accomplished by heatwelding.
 17. A method for treating a wound, comprising: a) providing; i)keratinocytes on a solid support, ii) an enclosure, said enclosurehousing said solid support, wherein said enclosure comprises meshmaterial having pores, said pores being too small to permit saidkeratinocytes on said solid support to cross said mesh material, andiii) a subject having a wound; and b) positioning said enclosure in saidwound of said subject under conditions such that healing of said woundis promoted.
 18. The method of claim 17, wherein said solid supportcomprises beads.
 19. The method of claim 18, wherein said beads comprisemacroporous beads.
 20. The method of claim 19, wherein said macroporousbeads comprise a collagen coating.
 21. The method of claim 17, furthercomprising step c) covering said enclosure with a dressing.